Rescuing the Last-Line Polymyxins: Achievements and Challenges.

Antibiotic resistance is a major global health challenge and, worryingly, several key Gram negative pathogens can become resistant to most currently available antibiotics. Polymyxins have been revived as a last-line therapeutic option for the treatment of infections caused by multidrug-resistant Gram negative bacteria, in particular Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterales. Polymyxins were first discovered in the late 1940s but were abandoned soon after their approval in the late 1950s as a result of toxicities (e.g., nephrotoxicity) and the availability of "safer" antibiotics approved at that time. Therefore, knowledge on polymyxins had been scarce until recently, when enormous efforts have been made by several research teams around the world to elucidate the chemical, microbiological, pharmacokinetic/pharmacodynamic, and toxicological properties of polymyxins. One of the major achievements is the development of the first scientifically based dosage regimens for colistin that are crucial to ensure its safe and effective use in patients. Although the guideline has not been developed for polymyxin B, a large clinical trial is currently being conducted to optimize its clinical use. Importantly, several novel, safer polymyxin-like lipopeptides are developed to overcome the nephrotoxicity, poor efficacy against pulmonary infections, and narrow therapeutic windows of the currently used polymyxin B and colistin. This review discusses the latest achievements on polymyxins and highlights the major challenges ahead in optimizing their clinical use and discovering new-generation polymyxins. To save lives from the deadly infections caused by Gram negative "superbugs," every effort must be made to improve the clinical utility of the last-line polymyxins. SIGNIFICANCE STATEMENT: Antimicrobial resistance poses a significant threat to global health. The increasing prevalence of multidrug-resistant (MDR) bacterial infections has been highlighted by leading global health organizations and authorities. Polymyxins are a last-line defense against difficult-to-treat MDR Gram negative pathogens. Unfortunately, the pharmacological information on polymyxins was very limited until recently. This review provides a comprehensive overview on the major achievements and challenges in polymyxin pharmacology and clinical use and how the recent findings have been employed to improve clinical practice worldwide.

Therapeutic Potential of Intravenous Phage as Standalone Therapy for Recurrent Drug-Resistant Urinary Tract Infections.

Recurrent urinary tract infections (rUTI) are common in kidney transplant recipients (KTR) and are associated with multidrug resistance and increased morbidity/mortality. Novel antibiotic alternatives to reduce UTI recurrence are critically needed. We describe a case of rUTI due to extended spectrum beta lactamase (ESBL) Klebsiella pneumoniae in a KTR that was treated successfully with 4 weeks of adjunctive intravenous bacteriophage therapy alone, without concomitant antibiotics, and with no recurrence in a year of follow-up.

Polymyxin resistance in Klebsiella pneumoniae: multifaceted mechanisms utilized in the presence and absence of the plasmid-encoded phosphoethanolamine transferase gene mcr-1.

OBJECTIVES: Until plasmid-mediated mcr-1 was discovered, it was believed that polymyxin resistance in Gram-negative bacteria was mainly mediated by the chromosomally-encoded EptA and ArnT, which modify lipid A with phosphoethanolamine (pEtN) and 4-amino-4-deoxy-l-arabinose (l-Ara4N), respectively. This study aimed to construct a markerless mcr-1 deletion mutant in Klebsiella pneumoniae, validate a reliable reference gene for reverse transcription quantitative PCR (RT-qPCR) and investigate the interactions among mcr-1, arnT and eptA, in response to polymyxin treatments using pharmacokinetics/pharmacodynamics (PK/PD). METHODS: An isogenic markerless mcr-1 deletion mutant (II-503Δmcr-1) was generated from a clinical K. pneumoniae II-503 isolate. The efficacy of different polymyxin B dosage regimens was examined using an in vitro one-compartment PK/PD model and polymyxin resistance was assessed using population analysis profiles. The expression of mcr-1, eptA and arnT was examined using RT-qPCR with a reference gene pepQ, and lipid A was profiled using LC-MS. In vivo polymyxin B efficacy was investigated in a mouse thigh infection model. RESULTS: In K. pneumoniae II-503, mcr-1 was constitutively expressed, irrespective of polymyxin exposure. Against II-503Δmcr-1, an initial bactericidal effect was observed within 4 h with polymyxin B at average steady-state concentrations of 1 and 3 mg/L, mimicking patient PK. However, substantial regrowth and concomitantly increased expression of eptA and arnT were detected. Predominant l-Ara4N-modified lipid A species were detected in II-503Δmcr-1 following polymyxin B treatment. CONCLUSIONS: This is the first study demonstrating a unique markerless deletion of mcr-1 in a clinical polymyxin-resistant K. pneumoniae. The current polymyxin B dosage regimens are suboptimal against K. pneumoniae, regardless of mcr, and can lead to the emergence of resistance.

The rise and spread of mcr plasmid-mediated polymyxin resistance.

Polymyxins are important lipopeptide antibiotics that serve as the last-line defense against multidrug-resistant (MDR) Gram-negative bacterial infections. Worryingly, the clinical utility of polymyxins is currently facing a serious threat with the global dissemination of mcr, plasmid-mediated polymyxin resistance. The first plasmid-mediated polymyxin resistance gene, termed as mcr-1 was identified in China in November 2015. Following its discovery, isolates carrying mcr, mainly mcr-1 and less commonly mcr-2 to -7, have been reported across Asia, Africa, Europe, North America, South America and Oceania. This review covers the epidemiological, microbiological and genomics aspects of this emerging threat to global human health. The mcr has been identified in various species of Gram-negative bacteria including Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella enterica, Cronobacter sakazakii, Kluyvera ascorbata, Shigella sonnei, Citrobacter freundii, Citrobacter braakii, Raoultella ornithinolytica, Proteus mirabilis, Aeromonas, Moraxella and Enterobacter species from animal, meat, food product, environment and human sources. More alarmingly is the detection of mcr in extended-spectrum-ß-lactamases- and carbapenemases-producing bacteria. The mcr can be carried by different plasmids, demonstrating the high diversity of mcr plasmid reservoirs. Our review analyses the current knowledge on the emergence of mcr-mediated polymyxin resistance.

Fitness cost of mcr-1-mediated polymyxin resistance in Klebsiella pneumoniae.

Objectives: The discovery of mobile colistin resistance mcr-1, a plasmid-borne polymyxin resistance gene, highlights the potential for widespread resistance to the last-line polymyxins. In the present study, we investigated the impact of mcr-1 acquisition on polymyxin resistance and biological fitness in Klebsiella pneumoniae. Methods: K. pneumoniae B5055 was used as the parental strain for the construction of strains carrying vector only (pBBR1MCS-5) and mcr-1 recombinant plasmids (pmcr-1). Plasmid stability was determined by serial passaging for 10 consecutive days in antibiotic-free LB broth, followed by patching on gentamicin-containing and antibiotic-free LB agar plates. Lipid A was analysed using LC-MS. The biological fitness was examined using an in vitro competition assay analysed with flow cytometry. The in vivo fitness cost of mcr-1 was evaluated in a neutropenic mouse thigh infection model. Results: Increased polymyxin resistance was observed following acquisition of mcr-1 in K. pneumoniae B5055. The modification of lipid A with phosphoethanolamine following mcr-1 addition was demonstrated by lipid A profiling. The plasmid stability assay revealed the instability of the plasmid after acquiring mcr-1. Reduced in vitro biological fitness and in vivo growth were observed with the mcr-1-carrying K. pneumoniae strain. Conclusions: Although mcr-1 confers a moderate level of polymyxin resistance, it is associated with a significant biological fitness cost in K. pneumoniae. This indicates that mcr-1-mediated resistance in K. pneumoniae could be attenuated by limiting the usage of polymyxins.

PhageGE: an interactive web platform for exploratory analysis and visualization of bacteriophage genomes.

BACKGROUND: Antimicrobial resistance is a serious threat to global health. Due to the stagnant antibiotic discovery pipeline, bacteriophages (phages) have been proposed as an alternative therapy for the treatment of infections caused by multidrug-resistant pathogens. Genomic features play an important role in phage pharmacology. However, our knowledge of phage genomics is sparse, and the use of existing bioinformatic pipelines and tools requires considerable bioinformatic expertise. These challenges have substantially limited the clinical translation of phage therapy. FINDINGS: We have developed PhageGE (Phage Genome Explorer), a user-friendly graphical interface application for the interactive analysis of phage genomes. PhageGE enables users to perform key analyses, including phylogenetic analysis, visualization of phylogenetic trees, prediction of phage life cycle, and comparative analysis of phage genome annotations. The new R Shiny web server, PhageGE, integrates existing R packages and combines them with several newly developed functions to facilitate these analyses. Additionally, the web server provides interactive visualization capabilities and allows users to directly export publication-quality images. CONCLUSIONS: PhageGE is a valuable tool that simplifies the analysis of phage genome data and may expedite the development and clinical translation of phage therapy. PhageGE is publicly available at https://jason-zhao.shinyapps.io/PhageGE_Update/.

Phage resistance in Klebsiella pneumoniae and bidirectional effects impacting antibiotic susceptibility.

OBJECTIVES: Bacteriophage (phage) therapy is a promising anti-infective option to combat antimicrobial resistance. However, the clinical utilization of phage therapy has been severely compromised by the potential emergence of phage resistance. Although certain phage resistance mechanisms can restore bacterial susceptibility to certain antibiotics, a lack of knowledge of phage resistance mechanisms hinders optimal use of phages and their combination with antibiotics. METHODS: Genome-wide transposon screening was performed with a mutant library of Klebsiella pneumoniae MKP103 to identify phage pKMKP103_1-resistant mutants. Phage-resistant phenotypes were evaluated by time-kill kinetics and efficiency of plating assays. Phage resistance mechanisms were investigated with adsorption, one-step growth, and mutation frequency assays. Antibiotic susceptibility was determined with broth microdilution and population analysis profiles. RESULTS: We observed a repertoire of phage resistance mechanisms in K pneumoniae, such as disruption of phage binding (fhuA::Tn and tonB::Tn), extension of the phage latent period (mnmE::Tn and rpoN::Tn), and increased mutation frequency (mutS::Tn and mutL::Tn). Notably, in contrast to the prevailing view that phage resistance re-sensitizes antibiotic-resistant bacteria, we observed a bidirectional steering effect on bacterial antibiotic susceptibility. Specifically, rpoN::Tn increased susceptibility to colistin while mutS::Tn and mutL::Tn increased resistance to rifampicin and colistin. DISCUSSION: Our findings demonstrate that K pneumoniae employs multiple strategies to overcome phage infection, which may result in enhanced or reduced antibiotic susceptibility. Mechanism-guided phage steering should be incorporated into phage therapy to better inform clinical decisions on phage-antibiotic combinations.

The Safety of Bacteriophages in Treatment of Diseases Caused by Multidrug-Resistant Bacteria.

Given the urgency due to the rapid emergence of multidrug-resistant (MDR) bacteria, bacteriophages (phages), which are viruses that specifically target and kill bacteria, are rising as a potential alternative to antibiotics. In recent years, researchers have begun to elucidate the safety aspects of phage therapy with the aim of ensuring safe and effective clinical applications. While phage therapy has generally been demonstrated to be safe and tolerable among animals and humans, the current research on phage safety monitoring lacks sufficient and consistent data. This emphasizes the critical need for a standardized phage safety assessment to ensure a more reliable evaluation of its safety profile. Therefore, this review aims to bridge the knowledge gap concerning phage safety for treating MDR bacterial infections by covering various aspects involving phage applications, including phage preparation, administration, and the implications for human health and the environment.

Pharmacokinetics/pharmacodynamics of phage therapy: a major hurdle to clinical translation.

BACKGROUND: The increasing emergence of antimicrobial resistance worldwide has led to renewed interest in phage therapy. Unlike antibiotics, the lack of pharmacokinetics/pharmacodynamics (PK/PD) information represents a major challenge for phage therapy. As therapeutic phages are biological entities with the ability to self-replicate in the presence of susceptible bacteria, their PK/PD is far more complicated than that of antibiotics. OBJECTIVES: This narrative review examines the current literature on phage pharmacology and highlights major pharmacological challenges for phage therapy. SOURCES: Included articles were identified by searching PubMed and Google Scholar till June 2022. The search terms were 'bacteriophage', 'antimicrobial', 'pharmacokinetics' and 'pharmacodynamics'. Additional relevant references were obtained from articles retrieved from the primary search. CONTENT: In this review, phage PK is first discussed, focusing on absorption, distribution, metabolism, and elimination. Key factors affecting phage antimicrobial activities are reviewed, including multiplicity of infection, passive and active phage therapy, and the involvement of the human immune system. Importantly, we emphasize the impact of phage self-replication on the PK/PD and the fundamental phage characteristics that are required for PK/PD modelling and clinical translation. IMPLICATIONS: Recent progress in phage pharmacology has shown that we are in a far better position now to treat infections with phage therapy than a century ago. However, phage therapy is still in its infancy when compared to antibiotics due to the scarce pharmacological knowledge (e.g. PK/PD). Optimization of phage PK/PD is key for translation of phage therapy in patients.

Greater Invasion and Persistence of mcr-1-Bearing Plasmids in Escherichia coli than in Klebsiella pneumoniae.

The emergence of the plasmid-borne polymyxin resistance gene mcr-1 threatens the clinical utility of last-line polymyxins. Although mcr-1 has disseminated to various Enterobacterales species, the prevalence of mcr-1 is the highest among Escherichia coli isolates while remaining low in Klebsiella pneumoniae. The reason for such a difference in prevalence has not been investigated. In this study, we examined and compared the biological characteristics of various mcr-1 plasmids in these two bacterial species. Although mcr-1-bearing plasmids were stably maintained in both E. coli and K. pneumoniae, the former presented itself to be superior by demonstrating a fitness advantage while carrying the plasmid. The inter- and intraspecies transferability efficiencies were evaluated for common mcr-1-harboring plasmids (IncX4, IncI2, IncHI2, IncP, and IncF types) with native E. coli and K. pneumoniae strains as donors. Here, we found that the conjugation frequencies of mcr-1 plasmids were significantly higher in E. coli than in K. pneumoniae, regardless of the donor species and Inc types of the mcr-1 plasmids. Plasmid invasion experiments revealed that mcr-1 plasmids displayed greater invasiveness and stability in E. coli than in K. pneumoniae. Moreover, K. pneumoniae carrying mcr-1 plasmids showed a competitive disadvantage when cocultured with E. coli. These findings indicate that mcr-1 plasmids could spread more easily among E. coli than among K. pneumoniae isolates and that mcr-1 plasmid-carrying E. coli has a competitive advantage over K. pneumoniae, leading to E. coli being the main mcr-1 reservoir. IMPORTANCE As infections caused by multidrug-resistant "superbugs" are increasing globally, polymyxins are often the only viable therapeutic option. Alarmingly, the wide spread of the plasmid-mediated polymyxin resistance gene mcr-1 is restricting the clinical utility of this last-line treatment option. With this, there is an urgent need to investigate the factors contributing to the spread and persistence of mcr-1-bearing plasmids in the bacterial community. Our research highlights that the higher prevalence of mcr-1 in E. coli than in K. pneumoniae is attributed to the greater transferability and persistence of mcr-1-bearing plasmid in the former species. By gaining these important insights into the persistence of mcr-1 in different bacterial species, we will be able to formulate effective strategies to curb the spread of mcr-1 and prolong the clinical life span of polymyxins.

Novel antimicrobial agents for combating antibiotic-resistant bacteria.

Antibiotic therapy has become increasingly ineffective against bacterial infections due to the rise of resistance. In particular, ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have caused life-threatening infections in humans and represent a major global health threat due to a high degree of antibiotic resistance. To respond to this urgent call, novel strategies are urgently needed, such as bacteriophages (or phages), phage-encoded enzymes, immunomodulators and monoclonal antibodies. This review critically analyses these promising antimicrobial therapies for the treatment of multidrug-resistant bacterial infections. Recent advances in these novel therapeutic strategies are discussed, focusing on preclinical and clinical investigations, as well as combinatorial approaches. In this 'Bad Bugs, No Drugs' era, novel therapeutic strategies can play a key role in treating deadly infections and help extend the lifetime of antibiotics.

Polymyxin causes cell envelope remodelling and stress responses in mcr-1-harbouring Escherichia coli.

Polymyxins remain important last-line antibiotics against multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance is emerging and the mobile polymyxin resistance gene, mcr, is contributing to the wide dissemination of polymyxin resistance, especially among Escherichia coli, with mcr-1 being the most commonly found variant. The objective of this study was to provide mechanistic insights into concentration-dependent transcriptomic responses of mcr-harbouring E. coli following polymyxin treatment. An mcr-1-carrying clinical isolate of E. coli (LH30) was treated with polymyxin B at 2 and 8 mg/L. Bacterial cultures were collected before and 1 h following treatment for viable counting and transcriptomic analysis. Growth of E. coli LH30 was unaffected by 2 mg/L polymyxin B, whereas killing of approximately 2 log10 colony-forming units/mL occurred with 8 mg/L at 1 h. All four phosphoethanolamine (pEtN) transferase genes (mcr-1, eptA, eptB and eptC) were upregulated (fold change 2.4-4.0) by 8 mg/L polymyxin B, indicating that pEtN modifications were the preferred polymyxin resistance mechanism. The higher polymyxin B concentration also affected the expression of genes involved in fatty acid, lipopolysaccharide, lipid A, phospholipid biosynthesis, iron homeostasis and oxidative stress pathways. This transcriptomic analysis revealed that cell envelope remodelling, pEtN modification, iron acquisition and oxidative stress protective mechanisms play a key role in the survival of mcr-carrying E. coli treated with polymyxin. These findings provide new mechanistic information at the gene expression level to counter polymyxin resistance.

Pharmacokinetics and pharmacodynamics of peptide antibiotics.

Antimicrobial resistance is a major global health challenge. As few new efficacious antibiotics will become available in the near future, peptide antibiotics continue to be major therapeutic options for treating infections caused by multidrug-resistant pathogens. Rational use of antibiotics requires optimisation of the pharmacokinetics and pharmacodynamics for the treatment of different types of infections. Toxicodynamics must also be considered to improve the safety of antibiotic use and, where appropriate, to guide therapeutic drug monitoring. This review focuses on the pharmacokinetics/pharmacodynamics/toxicodynamics of peptide antibiotics against multidrug-resistant Gram-negative and Gram-positive pathogens. Optimising antibiotic exposure at the infection site is essential for improving their efficacy and minimising emergence of resistance.

Comparative metabolomics revealed key pathways associated with the synergistic killing of multidrug-resistant Klebsiella pneumoniae by a bacteriophage-polymyxin combination.

Resistance to the last-line polymyxins is emerging in multidrug-resistant Klebsiella pneumoniae and phage therapy is a promising alternative. However, phage monotherapy often rapidly causes resistance and few studies have examined antibiotic-phage combinations against K. pneumoniae. Here, we investigated the combination of polymyxin B with a novel phage pK8 against an mcr-1-carrying polymyxin-resistant clinical isolate Kp II-503 (polymyxin B MIC, 8 mg/L). The phage genome was sequenced and bacterial metabolomes were analysed at 4 and 24 h following the treatment with polymyxin B (16 mg/L), phage pK8 (102 PFU/mL) and their combination. Minimal metabolic changes across 24 h were observed with polymyxin B alone; whereas a significant inhibition of the citrate cycle, pentose phosphate pathway, amino acid and nucleotide metabolism occurred with the phage-polymyxin combination at both 4 and 24 h, but with phage alone only at 4 h. The development of resistance to phage alone was associated with enhanced membrane lipid and decreased amino acid biosynthesis in Kp II-503. Notably, cAMP, cGMP and cCMP were significantly enriched (3.1-6.6 log2fold) by phage alone and the combination only at 4 h. This is the first systems pharmacology study to investigate the enhanced bacterial killing by polymyxin-phage combination and provides important mechanistic information on phage killing, resistance and antibiotic-phage combination in K. pneumoniae.

Metabolic Perturbations Caused by the Over-Expression of mcr-1 in Escherichia coli.

Rapid dissemination of the plasmid-born polymyxin resistance gene mcr-1 poses a critical medical challenge. MCR-1 expression is tightly controlled and imposes a fitness cost on the bacteria. We used growth studies and metabolomics to examine growth and metabolic changes within E. coli TOP10 at 8 and 24 h in response to different levels of expression of mcr-1. Induction of mcr-1 greatly increased expression at 8 h and markedly reduced bacterial growth; membrane disruption and cell lysis were evident at this time. At 24 h, the expression of mcr-1 dramatically declined with restored growth and membrane integrity, indicating regulation of mcr-1 expression in bacteria to maintain membrane homeostasis. Intermediates of peptide and lipid biosynthesis were the most commonly affected metabolites when mcr-1 was overexpressed in E. coli. Cell wall biosynthesis was dramatically affected with the accumulation of lipids including fatty acids, glycerophospholipids and lysophosphatidylethanolamines, especially at 8 h. In contrast, levels of intermediate metabolites of peptides, amino sugars, carbohydrates and nucleotide metabolism and secondary metabolites significantly decreased. Moreover, the over-expression of mcr-1 resulted in a prolonged reduction in intermediates associated with pentose phosphate pathway and pantothenate and CoA biosynthesis. These findings indicate that over-expression of mcr-1 results in global metabolic perturbations that mainly involve disruption to the bacterial membrane, pentose phosphate pathway as well as pantothenate and CoA biosynthesis.